The present invention relates to methods for identifying inhibitors of the bitter taste response, and by methods of using such inhibitors to either block the perception of bitterness and/or promote the perception of a sweet taste. The inhibitors of the invention may be used as flavor enhancers in foods and pharmaceuticals. The methods of the invention may further be used to characterize the gustatory perception of novel tastants.
The sensation of taste has profound biological significance. It has much wider ramifications than merely providing mankind with pleasurable culinary experiences. Taste conveys numerous biological cues to humans and other animals, identifying tainted or spoiled foods and providing hedonic responses which may be proportionate to caloric or nutritive value.
There are generally considered to be only four or five categories of basic taste: sweet, sour, bitter, acid, and xe2x80x9cumamixe2x80x9d (the Japanese word describing the taste of monosodium glutamate; Hemess, M. S. and Gilbertson, T. A., 1999, Annu. Rev. Physiol. 61:873-900). These can be sub-classified as the appetitive tastes, such as salty, sweet and umami, which are associated with nutrient-containing foods, and the bitter and sour tastes elicited by toxic compounds. The latter two produce an aversive reaction which may protect an organism by discouraging the ingestion of unhealthy or dangerous foods. Among the undesirable compounds associated with a bitter taste are plant alkaloids such as caffeine, strychnine and quinine, cyanide, and metabolic waste products such as urea (Lindemann, B., 1996, Physiol. Rev. 76:719-766). It has recently been suggested that fat, the most energy-dense nutrient, may possess gustatory cues (Id., citing Gilbertson T. A. et al., 1997, Am. J. Physiol. 272:C1203-1210 and Gilbertson, T. A., 1998, Curr. Opin. Neurobiol. 8:447-452).
The anatomic basis for the initial events of taste is the taste receptor cell (xe2x80x9cTRCxe2x80x9d), located in clusters referred to as xe2x80x9ctaste budsxe2x80x9d (Lindemann, supra). Taste buds are distributed throughout the oral cavity, including the tongue as well as extra-lingual locations (see Hemess and Gilbertson). In the human tongue, taste buds are organized into three specialized types of specialized structures, namely fungiform, foliate, and circumvallate papillae. Each taste bud comprises between about 50 and 100 individual cells grouped into a cluster that is between 20 and 40 microns in diameter. Nerve fibers enter from the base of the taste bud and synapse onto some of the taste receptor cells. Typically, a single TRC contacts several sensory nerve fibers, and each sensory fiber innervates several TRCs in the same taste bud (Lindemann, supra).
When a subject ingests a tastant, and that tastant encounters a taste receptor cell in the appropriate concentration, an action potential is produced which, via synapses with primary sensory neurons, communicates the signal registered by the receptor, via afferent nerves, to the appropriate region of the sensory cortex of the brain, resulting in the perception of a particular taste by the subject. Food appraisal can give rise to a hedonic response involving the activation of midbrain dopamine neurons (Lindemann, supra, citing Mirenowicz, J. and Schultz, W., 1996, Nature (London) 379:449-451) and the release of endogenous opiates (Lindemann, supra, citing Drenowski, A., et al., 1992, Physiol. Behav. 51:371-379; Dum, J. et al., 1983, Pharmacol. Biochem. Behav. 18:443-447).
Much research has been directed toward elucidating the physiology of taste. TRCs of most, if not all, vertebrate species possess voltage-gated sodium, potassium, and calcium ion channels with properties similar to those of neurons (Kinnamon, S. C. and Margolskee, R. F., 1996, Curr. Opin. Neurobiol. 6:506-513). Different types of primary tastes appear to utilize different types of transduction mechanisms, and certain types of tastes may employ multiple mechanisms which may reflect varying nutritional requirements amongst species (Kinnamon and Margolskee, supra). For example, in the hamster, acid taste is associated with the influx of protons through an amiloride-sensitive sodium ion channel (Id., citing Gilbertson, T. A. et al., 1993, Neuron 10:931-942), whereas in the mudpuppy, a proton block of potassium ion channels at the apical cell membrane is involved (Kinnamon and Margolskee, supra, citing Kinnamon, S. C. et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7023-7027). Salty taste is typically transduced via permeation of sodium ions through amiloride-sensitive sodium channels.
Sweet taste has been associated with a second messenger system which may differ depending upon whether the tastant is a natural or artificial sweetener, the former believed to utilize cAMP, the latter inositol trisphosphate (IP3; Hemess M. S. and Gilbertson, T. A., 1999, Annu. Rev. Physiol. 61:873-900). There is evidence that a membrane-bound receptor, such as that involved in the activation of Gs and adenylyl cyclase, may be involved in the perception of sweet tastes (Id.).
Bitter taste sensations are also thought to involve cAMP and IP3 (Kinnamon and Margolskee, supra). The bitter compound denatonium causes calcium ion release from rat TRCs and the rapid elevation of IP3 levels in rodent taste tissue (Id., citing Bernhardt, S J. et al., 1996, J. Physiol. (London) 490:325-336 and Akabas, M. H., et al., 1988, Science 242:1047-1050). Since denatonium cannot pass the cell membrane, it has been suggested that it may activate G-protein-coupled receptors, whereby the xcex1 and/or xcex2xcex3 G protein subunits would activate phospholipase C, leading to IP3 generation and the release of calcium ions (Kinnamon and Margolskee, supra).
In recent years, a taste-specific G protein termed xe2x80x9cgustducinxe2x80x9d, which is homologous to the retinal G protein, transducin, has been cloned and characterized (Id., citing McLaughlin, S. et al., 1992, Nature (London) 357:563-569). Mice in which the a gustducin gene has been knocked out exhibit diminished responses to certain bitter (and certain sweet) tastants, suggesting that gustducin may regulate the TRC IP3 response (Kinnamon and Margolskee, citing Wong, G. T. et al., 1996, Nature (London) 381:796-800). Introducing a wild-type rat xcex1-gustducin-encoding cDNA into xcex1-gustducin null mice restored their responsiveness to bitter and sweet compounds (Ming, D. et al., 1998, Proc. Natl. Acad. Sci. U.S.A. 95:8933-8938, citing Wong, G. T., et al., 1996, Cold Spring Harbor Symp. Quant. Biol. 61:173-184). Gustducin""s xcex3 subunit (xcex313) has recently been shown to mediate activation of phospholipase C in response to the bitter compound denatonium (Huang, L. et al., 1999, Nature Neurosci. 2:1055-1062).
Although it had been believed that rod and cone transducins were specific G proteins present only in photoreceptor cells of the vertebrate retina (Lochrie, M. A. et al., 1985, Science 228:96-99; Medynski, D. C. et al., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:4311-4315; Tanabe, T. et al., 1985, Nature (London) 315:242-245; Yatsunami K. and Khorana, H. G., 1985, Proc. Natl. Acad. Sci. U.S.A. 82:4316-4320), it was discovered that od transducin is also present in vertebrate taste cells, where it specifically activates a phosphodiesterase isolated from taste tissue (Ruiz-Avila, L. et al., 1995, Nature (London) 376:80-85). Using a trypsin-sensitivity assay, it was demonstrated that the bitter compound denatonium, in the presence of taste cell membranes, activates transducin but not the G-protein, Gi (Id.). This activation could be inhibited by a peptide derived from the C-terminal region of transducin, which competitively inhibits the rhodopsin-transducin interaction (Id. and Hamm, H. E., et al., 1988, Science 241:832-8359). Ruiz-Avila et al. (supra) proposed that transducin may be involved in bitter taste transduction via a cascade similar to that which occurs in visual perception, whereby a stimulated bitter receptor may activate taste-cell transducin, which in turn activates phosphodiesterase. The activated phosphodiesterase may then decrease levels of intracellular 3xe2x80x2,5xe2x80x2-cyclic nucleotides, and the resulting lower levels of cyclic nucleotides could lead to TRC depolarization by a mechanism referred to as xe2x80x9ccyclic-nucleotide-suppressible conductancexe2x80x9d(Id. citing Kolesnikov, S. and Margolskee, R. F., 1995, Nature (London) 376:85-88).
More recently, Ming, D. et al., 1998, Proc. Natl. Acad. Sci. U.S.A. 95:8933-8938 reported that both gustducin and transducin, in the presence of bovine taste cell membranes, were specifically activated by a number of bitter compounds, including denatonium, quinine, and strychnine. This activation was found to depend upon an interaction with the C-terminus of gustducin and required the presence of G-protein xcex2xcex3 subunits; it could be competitively inhibited by peptides derived from the sites of interaction of rhodopsin and transducin.
The present invention relates to methods for identifying compounds which inhibit the sensory perception of bitterness. It is based, at least in part, on the discovery that adenosine monophosphate (AMP) and related compounds inhibited the activation of transducin by bitter tastant-stimulated taste receptors, decreased neuronal stimulation by said tastants, and resulted in behavioral responses which indicate that the sensation of bitterness was greatly diminished.
Inhibitors of the invention may be used to decrease or abrogate the perception of bitterness of bitter tastants, in which capacity they are referred to as xe2x80x9cbitterness inhibitorsxe2x80x9d. In related embodiments, the present invention provides for methods of decreasing the perception of bitterness associated with a tastant by co-administering one or more bitterness inhibitors, and also provides for compositions comprising a bitter tastant and a bitterness inhibitor.
Inhibitors of the invention may also be found to convey a perception of sweetness when they are present with or without other tastants; in this capacity, they are referred to as xe2x80x9cinhibitor sweetenersxe2x80x9d. In various embodiments, the present invention provides for methods of creating the perception of sweetness, in which an inhibitor sweeter is administered to a subject, and also provides for compositions comprising inhibitor sweeteners.
The inhibitors of the invention may be used to enhance the flavor of foods, beverages, and pharmaceuticals by decreasing or eliminating bitter taste features. In addition to increasing food consumer satisfaction, inhibitors of the invention may also permit the incorporation, into foods and pharmaceuticals, of bitter tastants that improve shelf-life or nutritive value. The inhibitors of the invention could increase food intake in humans or livestock. Moreover, inhibitors of the invention could render medical procedures involving bitter compositions more palatable, and improve compliance in drug regimens involving bitter tastants, particularly when administered to children.
In further embodiments, the present invention provides for methods for identifying and/or characterizing bitter tastants which evoke taste responses similar to those of known bitter compounds. Non-toxic bitter compounds identified in this manner could be used as additives to provoke a desired aversive responsexe2x80x94for example, to discourage ingestion of compositions containing these compounds by children or animals.